1. β-adrenoceptors mediate relaxation of bladder detrusor smooth muscle. This study investigates the contribution of β3-adrenoceptors to relaxation of the pig urinary bladder. 2. Cell membranes were prepared from detrusor muscle of the pig bladder dome and competition experiments with [3H]-dihydroalprenolol (DHA), a non-selective β-adrenoceptor antagonist was used as a specific radioligand to determine the presence of β-adrenoceptor subtypes. In functional experiments, isolated detrusor muscle strips were used to determine the potency of agonists and the affinity of antagonists. 3. In competition binding experiments, CGP20712A (β1-adrenoceptor selective) displaced [3H]-DHA from a single binding site with a low affinity. In contrast, displacement data for ICI 118551 (β2-adrenoceptor antagonist) and SR59230A (β3-adrenoceptor antagonist) best fitted a two-site model suggesting a predominant (70%) population of β3-adrenoceptors. 4. In functional studies, isoprenaline and salbutamol (β2-adrenoceptor agonist) relaxed KC1 precontracted muscle strips with high potency (pEC50 7.7 and 7.2, respectively), whilst CGP12177 and BRL37344 (β3-adrenoceptor agonists) had low potency and were partial agonists. CGP20712A and atenolol (β1-adrenoceptor antagonists) antagonised responses with a low affinity. ICI118551 antagonized responses to isoprenaline and salbutamol with a high affinity (pKB=7.8 and 8.7, respectively), but the Schild slopes were low suggesting that responses were mediated by more than one β-adrenoceptor. The Schild plot for SR59230A was biphasic, apparent pKB values for 3-10 nM SR59230A being 8.6 and those for 30 nM-1 μM being 7.7. 5. These data suggest that β3-adrenoceptors are the predominant β-adrenoceptor subtype present in the pig bladder and that β-adrenoceptor mediated responses of this tissue are mediated via both the β2- and β3-adrenoceptor subtypes.